Techniques in molecular and cellular biology often involve the introduction of an exogenous genetic sequence into a cell, for example encoding an open reading frame, and the expression of the exogenous sequence within the cell to generate the encoded peptide or protein. Typically, the open reading frame is incorporated into a suitable vector under the control of a promoter sequence and promoter elements suitable for the binding of appropriate transcription factors and RNA polymerase. In the way, the vector utilizes the expression machinery of the host cell to co-ordinate expression.
Such expression systems may be broadly divided into those that involve constitutive expression wherein the degree of expression cannot be controlled, and those that enable inducible expression wherein the degree of expression can be controlled by external factors. Inducible expression systems are particularly useful where it is desirable to carefully control expression of a foreign protein in a host cell, for example to simulate a normal, endogenous expression profile. In other circumstances, inducible expression may allow the study of proteins whose constitutive expression might not be tolerated by the host cell. In some circumstances, the only way to generate a cell line or a recombinant viral vector that expresses this protein, is to use an inducible system, which is maintained in the off state at most times, such that expression is turned on to a desired level, or for a desired period, only at the time of the experiment.
Some expression systems of the prior art are derived from prokaryotes, and of these two have proved particularly useful: namely the lac and the tet operons. More recently, another inducible system has been developed as disclosed in United States patent publication 2004/0205834 published Oct. 14, 2004, corresponding to U.S. application Ser. No. 10/135,362, filed 31 May 2002, which is incorporated herein by reference. In preferred embodiments, this system is suitable for use in mammalian cells for a range of applications including those that require tight control or the need high-level expression. This new gene-switch is similar to the widely used Tet-system(1), but it makes use of a different bacterial repressor derived from the Pseudomonas putida F1 p-cymene operon. In P. putida, the degradative pathway for p-cymene to its benzoate derivative p-cumate consists of 6 genes organized in an operon (cym). The cym operon is followed by the cmt operon that is responsible for the further degradation of cumate. The expression of the genes in both operons is regulated by a repressor protein molecule (CymR) of about 28 kD that binds operator sequences downstream of the start site of the promoter(2). CymR is in a DNA-binding configuration only in the absence of cymene or cumate, the effector molecule(3A, B).
United States patent publication 2004/0205834 discloses the transformation of CymR into an activator (cTA) by fusion to a VP16 activation domain. In the context of adenoviral vectors, relatively low amounts of AdCMVcTA are required to achieve high expression levels from the Cumate-regulated promoter CR5. This contrasts to the Tet-switch where large amounts of reporter and activator virus (AdCMVtTA) are required to achieve the same levels of activation. Furthermore, the CR5/cTA system performs better than the strong promoter CMV5 in all cell lines tested. Thus, cell lines stably expressing cTA are useful for achieving maximal protein expression. Since maximal protein production is presumably incompatible with maximal cell growth, the ability to down-regulate protein production in cTA cells in the presence of cumate during clone selection facilitates the selection of high producing clones. However, in its current configuration, this system is not as readily amenable to large-scale production since the induction of protein secretion requires the removal of cumate, a process that is cumbersome in large-scale culture.
There remains a continuing need for inducible expressions system that permit tight regulation of expression of associated exogenous sequences. In particular, there is an increasing need for inducible expression systems that permit regulation of the level and/or duration of expression. Ideally, such expression systems may permit near total silencing of gene expression as desired.